KR0154499B1 - Process for the preparation of of4949ñœ - Google Patents
Process for the preparation of of4949ñœInfo
- Publication number
- KR0154499B1 KR0154499B1 KR1019950042463A KR19950042463A KR0154499B1 KR 0154499 B1 KR0154499 B1 KR 0154499B1 KR 1019950042463 A KR1019950042463 A KR 1019950042463A KR 19950042463 A KR19950042463 A KR 19950042463A KR 0154499 B1 KR0154499 B1 KR 0154499B1
- Authority
- KR
- South Korea
- Prior art keywords
- of4949ii
- aspergillus niger
- medium
- strain
- present
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 6
- 238000002360 preparation method Methods 0.000 title description 2
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 230000003068 static effect Effects 0.000 claims abstract description 4
- 230000001093 anti-cancer Effects 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 239000003456 ion exchange resin Substances 0.000 claims description 2
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 230000001900 immune effect Effects 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 6
- 235000013402 health food Nutrition 0.000 abstract description 5
- 230000000975 bioactive effect Effects 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 239000000284 extract Substances 0.000 description 5
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- 239000013028 medium composition Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000519854 Talaromyces rugulosus Species 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 229910052805 deuterium Inorganic materials 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 2
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002766 immunoenhancing effect Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 1
- AXZJHDNQDSVIDR-NSHDSACASA-N 4178-93-2 Chemical compound CC(C)C[C@H](N)C(=O)NC1=CC=C([N+]([O-])=O)C=C1 AXZJHDNQDSVIDR-NSHDSACASA-N 0.000 description 1
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 238000005361 D2 NMR spectroscopy Methods 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- MBMLMWLHJBBADN-UHFFFAOYSA-N Ferrous sulfide Chemical compound [Fe]=S MBMLMWLHJBBADN-UHFFFAOYSA-N 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/208—Fungi extracts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
Abstract
본 발명은 생리활성 물질인 OF4949Ⅱ의 제조방법에 관한 것으로, 본 발명의 아스페르질러스 나이거(Aspergillus niger) F-580(KCTC8686P)를 정치배양하여 형성된 균사 덩어리로부터 OF4949Ⅱ를 분리하는 것을 특징으로 하는 OF4949Ⅱ의 제조방법에 따르면 우수한 건강식품 소재로 사용할 수 있는 OF4949Ⅱ를 제조할 수 있다.The present invention relates to a method for preparing a bioactive substance OF4949II, characterized in that the OF4949II is isolated from the mycelial mass formed by static culture of Aspergillus niger F-580 (KCTC8686P) of the present invention. According to the manufacturing method of OF4949Ⅱ, OF4949II which can be used as an excellent health food material can be manufactured.
Description
제1도는 OF4949Ⅱ를 생성하는 균주의 전자현미경 사진과 포자의 전자현미경 사진을 나타낸 것이다.Figure 1 shows the electron micrograph and the electron micrograph of the spores of the strain producing OF4949II.
본 발명은 생리활성 물질인 OF4949Ⅱ의 제조방법에 관한 것으로, 보다 상세하게는 아스페르질러스 나이거 F-580을 정치 배양하여 OF4949Ⅱ를 제조하는 방법에 관한 것이다.The present invention relates to a method for producing OF4949II, which is a bioactive substance, and more particularly, to a method for producing OF4949II by static culture of Aspergillus Niger F-580.
OF4949Ⅱ는 곰팡이의 일종인 페니실리움 루굴로섬(Penicillium rugulosum) OF4949Ⅱ 균주에서 생산되는 생리활성 물질로, 에리크(Ehrlich) 복수암종 세포의 아미노펩티데이스 B군의 활성을 강력하게 저해하고 지연형 과민성반응(delyed-type hypersensitivity)을 증가시킴으로서 면역활성 증가능을 가지며, 300mg/kg을 복강내에 투여하여도 무독한 것으로 알려져 있다(Sano, S. et al., Journal of Antibiotics, 39(12) 1674-1684(1986)). 또한 OF4949Ⅱ의 고형 형태의 육암종(carcinoma)에 대해 0.5 내지 5.0mg/kg/day에서 항종양 활성을 나타내며 5mg/kg/day을 투여하면 루이스(Lewis) 페암종의 전이를 억제하는 것으로 나타나 있는데, 이와 같은 항암활성 작용은 세포독성에 기인하지 않고 면역반응의 증가에 기인하는 것으로 알려져 있다(Sano, S. et al., Journal of Antibiotics, 40(4), 519-524(1986)).OF4949Ⅱ is a physiologically active substance produced by the fungus Penicillium rugulosum OF4949II strain, which strongly inhibits the activity of the aminopeptides B group in Ehrlich ascites carcinoma cells and delays hypersensitivity reactions. Increasing delyed-type hypersensitivity increases immune activity and is known to be innocuous even when administered intraperitoneally at 300 mg / kg (Sano, S. et al., Journal of Antibiotics, 39 (12) 1674-1684) 1986)). In addition, it showed antitumor activity at the solid form of carcinoma of OF4949II at 0.5 to 5.0 mg / kg / day and administration of 5 mg / kg / day inhibited the metastasis of Lewis carcinoma. Such anticancer activity is known not to be due to cytotoxicity but to an increase in immune response (Sano, S. et al., Journal of Antibiotics, 40 (4), 519-524 (1986)).
이와 같이 유용한 생리활성 물질로 사용 가능성이 있는 OF4949Ⅱ를 대량으로 발효생산 및 분리하기 위한 연구가 진행되고 있다. 즉 OF4949Ⅱ는 페니실리움 루굴로섬을 발효조에서 8일동안 배양하고 11가지의 단계를 거쳐 분리하는 방법에 의해 제조되고 있으므로(Sano, S. et al., Journal of Antibiotics, 39(12) 1674-1684 (1986)), 보다 간편하게 단축된 분리 경제방법과 에너지 절약형 발효방법이 요구되어지고 있다.As such, studies are being conducted to ferment and produce large quantities of OF4949II, which may be used as a useful bioactive substance. That is, OF4949II is prepared by incubating penicillium rugulosum in fermenter for 8 days and separating it through 11 steps (Sano, S. et al., Journal of Antibiotics, 39 (12) 1674-1684 (1986), a simpler method of economic separation and energy-saving fermentation methods are required.
최근에는 특정활성을 가지는 건강식품소재를 다양한 제료에서 생화학적 수단에 의해 탐색작업을 수행하고 있는데, 특히 미국식품의약국에서 안전하다고 인정된(GRAS급) 미생물 균체인 바실러스, 아스페르질러스 등을 탐색재로 많이 사용하고 있다. 따라서 이들 균에내에서 OF4949Ⅱ와 같은 면역증강 항암물질을 대량 생산할 수 있다면 우수한 건강식품 소재로 이용할 수 있을 것이라 기대된다.In recent years, health food materials with specific activities have been searched for by various chemicals and biochemical means. Especially, Bacillus and Aspergillus microorganisms recognized as safe (GRAS) by the US Food and Drug Administration It is widely used as a search material. Therefore, if these bacteria can produce large amounts of immuno-enhancing anti-cancer substances such as OF4949Ⅱ, it is expected to be used as an excellent health food material.
본 발명자들은 아미노펩티데이즈 저해물질을 개발하기 위해 계속 연구하던 중 페니실리운속 곰팡이가 아닌 GRAS급의 아스페르질러스 나이거(Aspergillus niger)에 속하는 곰팡이 균주가 정치배양에 의해 형성된 매트에서만 OF4949Ⅱ를 생산한다는 사실을 발견하여 이를 보다 간단히 분리정제하여 건강식품 소재 등으로 이용할 수 있도록 함으로써 본 발명의 완성에 이르게 되었다.The inventors of the present invention, while continuing to develop an aminopeptide inhibitor, produced OF4949II only in mats in which a fungal strain belonging to Aspergillus niger, a GRAS class, but not penicillium fungus, was formed by political culture. Discovering the fact that it can be more easily separated and purified to be used as a health food material, etc. has led to the completion of the invention.
본 발명의 목적은 면역증강 항암물질로 OF4949Ⅱ를 안전한 GRASR급 미생물로부터 간단하게 제조하는 방법을 제공하는 것이다.SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing OF4949II from a safe GRASR-class microorganism as an immune enhancing anticancer substance.
상기 목적을 달성하기 위해, 본 발명에서는 아스페르질러스 나이거(Aspergillus niger) F-580(KCTC 8686P)를 정치배양하여 형성된 균사 덩어리로부터 OF4949Ⅱ를 분리하는 것을 특징으로 하는 OF4949Ⅱ의 제조방법을 제공한다.In order to achieve the above object, the present invention provides a method of manufacturing OF4949II, characterized in that the OF4949II is separated from the mycelial mass formed by static culture of Aspergillus niger (Aspergillus niger) F-580 (KCTC 8686P). .
이하, 본 발명을 좀더 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명에 따라 OF4949Ⅱ를 생산하는 아스페르질러스 나이거 F-580 균주는 부엽토를 감자 포도당 한천배지(감자 20g, 포도당 20g, 한천 20g, 증류수 1ℓ)상에 놓아 27℃의 항온기에서 5일간 배양한 후 생성된 곰팡이의 포자를 상기 배지에 백금이로 접종하여 배양한 후 다시 상기 배지에 접종하는 조작을 반복하여 순수하게 토양으로부터 분리한 균주로서, 아미노펩티데이즈를 강력하게 저해하는 특성이 있으며 여러가지 배양학적 및 형태학적 특성에 의하여 아스페르질러스 나이거로 동정하였다.Aspergillus Niger F-580 strain producing OF4949II according to the present invention was placed on the potato glucose agar medium (potato 20g, glucose 20g, agar 20g, distilled water 1L) incubated for 5 days in a thermostat at 27 ℃ After the fungus spores were inoculated with platinum on the medium and incubated again, the strain was inoculated into the medium again and purely separated from the soil, and strongly inhibited aminopeptides. Aspergillus Niger was identified by morphological and morphological characteristics.
아스페르질러스 나이거 F-580 균주는 다음과 같은 배양 특징을 갖는다.Aspergillus Niger F-580 strain has the following culture characteristics.
(1) 차펙한천 배지에서의 생육상태(1) Growth status in Chapek agar medium
·배지 조성 : 질산나트륨 3g, 인산수소이칼륨 1g, 황산마그네슘 0.5g, 염화칼슘 0.5g, 황화철 0.01g, 설탕 30g, 한천 20g, 중류수 1ℓMedium composition: Sodium nitrate 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, calcium chloride 0.5g, iron sulfide 0.01g, sugar 30g, agar 20g, middle water 1ℓ
·상기 배지에서 배양시 조밀한 황백색의 균사체와 흑색의 분생자 부위로 구성되고 포자를 많이 생성한다. 배면은 노란색으로 주름진 형태로 관찰된다.When cultured in the medium, it consists of dense yellowish white mycelium and black conidia. Back view as yellow corrugated.
(2) 맥아추축물 한천배지에서의 생육상태(2) Growth status on malt agar medium
·배지 조성 : 맥아추축물 20g, 한천 20g, 중류수 1ℓMedium composition: malt extract 20g, agar 20g, middle water 1ℓ
·상기 배지에서 배양시 균총이 넓고 조밀하지 않게 퍼지며 포자를 많이 형성한다. 흑색의 분생자 구조와 담황색의 배면이 관찰된다.When cultured in the medium, the flora is wide and dense spreads and forms a lot of spores. Black conidia structure and pale yellow back.
(3) 감자포도당 한천배지에서의 생육상태(3) Growth status on potato glucose agar medium
·배지조성 : 감자 20g, 포도당 20g, 한천 20g, 중류수 1ℓ· Medium composition: Potato 20g, Glucose 20g, Agar 20g, Mid-water 1ℓ
·상기 배지에서 배양시 균총이 넓고 조밀하게 퍼지며 잘 자라고 포자형성도 왕성하다. 분생자 부위의 색깔은 검은색이고 배면은 노란색이다. 분생자의 머리부위는 크고 방사상이며 후에 기둥형으로 갈라진다. 분생자병은 표면은 매끈하고 길이는 다양하며 지름은 13 내지 16㎛이다. 3내지 7×3 내지 4㎛의 경자(phialide)가 매튤래(metulae) 위에 형성되고 메튤래의 크기는 12 내지 20×4 내지 5㎛이다. 분생포자의 형태는 공모양이고 크기는 3내지 4㎛로 울퉁불퉁한 표면을 갖는다.When cultured in the medium, the flora is wide and densely spread, grows well, and spore formation is vigorous. The conidia area is black and the back is yellow. The head of the conidia is large and radial and later split into columns. Conidia disease has smooth surfaces, varying lengths, and diameters of 13-16 μm. A phialide of 3 to 7 x 3 to 4 mu m is formed on the matulae and the size of the tuple is 12 to 20 x 4 to 5 mu m. Conidia are ball-shaped and have a rugged surface with a size of 3 to 4 μm.
생육온도 범위는 15 내지 35℃이고 최적 온도는 25℃이며, 생육 pH 범위는 3 내지 8이고 최적 pH는 5내지 6이다.The growth temperature range is 15-35 ° C., the optimum temperature is 25 ° C., the growth pH range is 3-8, and the optimum pH is 5-6.
이상과 같이 분리 동정된 아스페르질러스 나이거 F-580 균주는 1995년 9월 4일자로 한국과학기술연구원 생명공학연구소 부설 유존자은행에 기탁번호 제 KCTC 8686P 호로서 기탁하였다.Aspergillus Niger F-580 strain isolated as described above was deposited on September 4, 1995, as a deposit No. KCTC 8686P to the resident bank attached to the Biotechnology Research Institute of the Korea Institute of Science and Technology.
상기 아스페르질러스 나이거 F-580 균주를 통상의 영양 배지에서 25 내지 30℃로 5 내지 7일간 정치배양하여 배양액 표면에 형성된 균사 덩어리를 열수추출하고, 이어서 이온교환수지 및 크로마토그래피 등 통상의 분리수단을 적절히 조합하여 OF4949Ⅱ를 분리정제할 수 있다. 순수하게 분리정제된 OF4949Ⅱ는 분자량, 자외선 흡수스펙트럼 및 증수소핵자기 공명스펙트럼 등을 사용하여 확인할 수 있다.The Aspergillus Niger F-580 strain was incubated in a conventional nutrient medium at 25 to 30 ° C. for 5 to 7 days to hydrothermally extract the mycelial lumps formed on the surface of the culture medium, followed by conventional ion exchange resins and chromatography. OF4949II can be separated and purified by appropriate combination of separation means. Purely purified OF4949II can be identified using molecular weight, ultraviolet absorption spectrum and distillation nuclear magnetic resonance spectrum.
OF4949Ⅱ는 본 발명의 아스페르질러스 나이거 F-580 균주를 정치배양시 배양액 표면에 형성되는 균사 덩어리에만 존재하며, 진탕배양시에는 생산되지 않는다.OF4949II is present only in the mycelial mass formed on the surface of the culture medium of the Aspergillus Niger F-580 strain of the present invention, it is not produced during shaking culture.
이하 본 발명은 하기 실시예에 의해 설명한다. 단, 하기 실시예는 본 발명을 보다 상세하게 설명하기 위한 것이며, 본 발명의 범위가 이들만으로 제하되는 것은 아니다The invention is illustrated by the following examples. However, the following Examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited only to these.
[실 시 예 1][Example 1]
OF4949Ⅱ의 제조 및 분리정제Preparation and purification of OF4949Ⅱ
아스페르질러스 나이거 F-580 균주(KCTC 8686P)를 탄소원으로는 포도당, 설탕, 전분을 하용하고 질소원으로는 대두박, 어분, 펩톤 등 통상의 미생물이 이용하는 영양원을 함유하는 배지(필요에 따라서는 나트륨, 칼륨, 염소, 인산 등을 첨가하기도 한다)에서 5 내지 7일 동안 25 내지 30℃에서 정치배양하였다. 배양액 표면에 형성된 균사 덩어리를 원래 배지량과 동일한 증류수에 넣어 120℃, 1기압하에서 5분간 처리하고 거즈로 걸러 균사 덩어리의 열수추출물을 얻었다. 상기 열수추출물은 다이아이온 HP20(Diaion HP20)에 흡착시켜 30% 메탄올로 용출시키고, 용출액을 DEAE 셀룰로즈에 흡착시켜 0.1% 아세트산으로 용출시켜 감압건조하고 소량의 물에 녹인 후, 세파덱스 G-10에서 물로 겔여과크로마토그래피를 수행하였다. 활성분획을 농축하여 YMC-ODS-AQ 칼럼에서 15% 메탄올로 용매로 고속액체 크로마토그래피하여 최종적으로 OF4949Ⅱ를 순수하게 분리정재하였다.Aspergillus Niger F-580 strain (KCTC 8686P) is a medium containing glucose, sugar and starch as a carbon source, and a medium containing nutrients used by ordinary microorganisms such as soybean meal, fish meal and peptone as nitrogen sources (if necessary Sodium, potassium, chlorine, phosphoric acid, etc. may be added) and incubated at 25-30 ° C. for 5-7 days. The mycelial lumps formed on the surface of the culture solution were placed in the same distilled water as the original medium, and treated at 120 ° C. and 1 atm for 5 minutes, and filtered to obtain hot water extracts of the mycelial lumps. The hot water extract was eluted with Diaion HP20, eluted with 30% methanol, the eluted solution was adsorbed with DEAE cellulose, eluted with 0.1% acetic acid, dried under reduced pressure and dissolved in a small amount of water, followed by Sephadex G-10. Gel filtration chromatography was performed with water. The active fractions were concentrated and subjected to high performance liquid chromatography with 15% methanol on a YMC-ODS-AQ column in high solvent to finally separate and purified OF4949II purely.
액체배지를 1㎝ 깊이로 만들어 접종하여 배양하였을시 형성된 균사 덩어리로부터는 1ℓ당 2.3㎎의 OF4949Ⅱ를 분리할 수 있었다.2.3mg of OF4949II per liter could be isolated from the mycelial masses formed when the liquid medium was inoculated with a depth of 1 cm.
OF4949Ⅱ의 배양공정이나 분리정제 공정에서의 추적은 다음과 같이 항아미노펩티데이즈 M 활성의 측정에 기준을 두어 행하였다. 즉, 96웰 마이크로플레이트(microplate)에 배양액 또는 물에 녹인 검체를 넣고, L-로이신-p-니트로아닐리드 저장액(12,5㎎/디메틸설폭사이드 1㎖)Tracking of the OF4949II in the culturing step and the separation and purification step was performed based on the measurement of antiaminopeptide M activity as follows. That is, put a sample dissolved in culture or water in a 96-well microplate (L, leucine-p-nitroanilide stock solution (12,5mg / dimethyl sulfoxide 1ml)
100㎕를 0.1M 트리염산완충용액(pH 7.0) 10㎖에 사용직전 희석하여 만든 용약 160㎕을 넣은 후, 돼지 췌장 유래의 아미노펩티데이즈 M 용액(1㎎/㎖) 3㎕를 첨가하여 30분간 반응시킬 때, 450nm로 마치크로플레이트리더(바이오라드사)에서 흡광도를 측정하여 색깔이 노랗게 변하지 않는 부위에 아미노펩티데이즈 M 활성의 저해물질, 즉 OF4949Ⅱ가 존재하는 것으로 하였다.Add 100 µl of the diluted solution immediately before use to 10 ml of 0.1 M trichloric acid buffer solution (pH 7.0), and add 3 µl of aminopeptides M solution (1 mg / ml) derived from pig pancreas for 30 minutes. When reacting, the absorbance was measured at 450 nm by a macroplate reader (Biorad Co., Ltd.), and it was assumed that an inhibitor of aminopeptide M activity, ie, OF4949II, was present at a site where the color did not turn yellow.
[실 시 예 2][Example 2]
OF4949Ⅱ의 구조동정Structural Identification of OF4949Ⅱ
아스페르질러스 나이거 F-580에서 분리 정제된 물질이 OF4949Ⅱ인지를 확인하기 위하여 중수소핵자기 공명스펙트럼, 자외선 흡수스펙트럼 및 분자량을 측정하였다. 아스페르질러스 나이거 F-580에서 분리정제된 물질은 이온스프레이 질량분석기에서 측정한 결과 m.w. 472로 기지의 OF4949Ⅱ 분자량과 동일하였으며 자외선 흡수스펙트럼은 OF4949Ⅱ와 마찬가지로 중성용액에서는 209,272,279nm, 알칼리성 용액에서는 바소크로믹 이동(bathochromic shift)이 일어나 218,243,297nm에서 최대흡수파장을 나타내었다. 다음 표1은 아스페르질러스 나이거 F-580 균주가 생성하는 아미노펩티데이즈 M 저해물질과 OF4949Ⅱ와의 중수소 NMR 스펙트럼을 비교하여 나타내었다. 표1에서 보듯이, 본 발명에 따라 제조된 아미노펩티데이즈 M 저해물질과 OF4949Ⅱ는 분자량, 자외선 흡수스펙트럼 및 중수소핵자기 공명스펙트럼이 동일하였으며, 이들은 동일 화합물임을 알 수 있었다.(S. Sano S et al., Journal of Antibiotics, 39(12), 1674-1684(1986))Deuterium nuclear magnetic resonance spectra, ultraviolet absorption spectra, and molecular weights were measured to determine whether the material purified from Aspergillus Niger F-580 was OF4949II. The purified material from Aspergillus Niger F-580 was measured by ion spray mass spectrometer. The molecular weight of 472 was the same as the known OF4949Ⅱ molecular weight, and the UV absorption spectrum showed the maximum absorption wavelength at 218,243,297nm with 209,272,279nm in neutral solution and bathochromic shift in alkaline solution. Table 1 shows the deuterium NMR spectra of the aminopeptides M inhibitor produced by Aspergillus Niger F-580 strain and OF4949II. As shown in Table 1, the aminopeptides M inhibitor prepared according to the present invention and OF4949II had the same molecular weight, ultraviolet absorption spectrum, and deuterium nuclear magnetic resonance spectrum, and they were found to be the same compound (S. Sano S et. al., Journal of Antibiotics, 39 (12), 1674-1684 (1986)).
[실 시 예 3][Example 3]
아스페르질러스 나이거 F-580 배양물중의 OF4949Ⅱ의 위치Location of OF4949Ⅱ in Aspergillus Niger F-580 Culture
포도당 1g, 가용성 전분 2g, 대두박 2.3g, 소금 0.2g, 효모추출물 0.4g 을 100㎖의 증류수에 녹여 1ℓ의 삼각플라스트에 넣고 면전을 한 후 120℃에서 15분간 멸균한 배지에, 아스페르질러스 나이거 F-580 균주를 접종하여 7일동안 정치배양하였다. 배양액의 표면에 형성된 곰팡이 균사덩어리를 수세하여 원래 배양액량과 동일한 량의 증류수에 넣고 120℃, 1 기압에서 5분간 처리하여 거즈로 거른 균사 덩어리의 열수추출물과 배양액의 항아미노펩티데이즈의 역가를 측정하였다. 그 결과, 저해역가는 균사덩어리의 열수추출물에서만 존재하는 것을 볼 수 있었다. 또한 동일한 균주를 동일한 조건에서 진탕배양하였을 경우에는 항아미노펩티데이즈 역가는 배양여액 및 군체에서 모두 존재하지 않았다.1 g of glucose, 2 g of soluble starch, 2.3 g of soybean meal, 0.2 g of salt, and 0.4 g of yeast extract are dissolved in 100 ml of distilled water and placed in 1 L of Erlenmeyer Plasma. Sniger F-580 strains were inoculated and incubated for 7 days. The fungal mycelium formed on the surface of the culture solution was washed with water and placed in the same amount of distilled water as the original culture solution and treated for 5 minutes at 120 ° C. and 1 atmosphere to measure the titer of the hot water extract of the mycelia mass filtered with gauze and the antiaminopeptides of the culture solution It was. As a result, it was found that the inhibitory titers exist only in the hydrothermal extracts of the mycelia. In addition, when the same strain was shaken under the same conditions, the antiaminopeptide titers were not present in both the culture filtrate and the colonies.
이상에서 살펴본 바와 같이 아스페르질러스 나이거 F-580 균주를 정치배양하고 균사 덩어리로부터 OF4949Ⅱ를 분리정제하는 본 발명의 방법에 따르면, 면역증강 항암활성이 있는 생리 활성물질 2.3㎎/1ℓ 배지의 양으로 생산할 수 있으며 이는 우수한 건강식품 소재로 이용될 수 있다.As described above, according to the method of the present invention for culturing the Aspergillus Niger F-580 strain and isolating and purifying OF4949II from the mycelium mass, the amount of the biologically active substance 2.3 mg / 1 L medium having immuno-enhancing anticancer activity It can be used as a good health food material.
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1019950042463A KR0154499B1 (en) | 1995-11-21 | 1995-11-21 | Process for the preparation of of4949ñœ |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR0154499B1 (en) |
-
1995
- 1995-11-21 KR KR1019950042463A patent/KR0154499B1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| KR970027293A (en) | 1997-06-24 |
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